Urothelium-Specific Expression of Mutationally Activated Pik3ca Initiates Early Lesions of Noninvasive Bladder Cancer.

Although approximately 70% of bladder cancers are noninvasive and have high recurrence rates, early-stage disease is understudied. The lack of models to validate the contribution of molecular drivers of bladder tumorigenesis is a significant issue. Although mutations in PIK3CA are frequent in human bladder cancer, an in vivo model for understanding their contribution to bladder tumorigenesis is unavailable. Therefore, a Upk2-Cre/Pik3caH1047R mouse model expressing one or two R26-Pik3caH1047R alleles in a urothelium-specific manner was generated. Pik3caH1047R functionality was confirmed by quantifying Akt phosphorylation, and mice were characterized by assessing urothelial thickness, nuclear atypia, and expression of luminal and basal markers at 6 and 12 months of age. While at 6 months, Pik3caH1047R mice developed increased urothelial thickness and nuclear atypia, progressive disease was not observed at 12 months. Immunohistochemistry showed urothelium maintained luminal differentiation characterized by high forkhead box A1 (Foxa1) and peroxisome proliferator-activated receptor γ expression. Surprisingly, Pik3caH1047R mice subjected to low-dose carcinogen exposure [N-butyl-N-(4-hydroxybutyl)nitrosamine] exhibited no significant differences after exposure relative to mice without exposure. Furthermore, single-sample gene set enrichment analysis of invasive human tumors showed those with mutant PIK3CA did not exhibit significantly increased phosphatidylinositol 3-kinase/AKT pathway activity compared with wild-type PIK3CA tumors. Overall, these data suggest that Pik3caH1047R can elicit early tumorigenic changes in the urothelium, but progression to invasion may require additional genetic alterations.

Clinicopathological risk factors associated with tumor relapse of upper tract urothelial carcinoma after radical nephroureterectomy: A single institution 20-year experience.

Upper tract urothelial carcinoma (UTUC) is a relatively rare yet aggressive malignancy. While radical nephroureterectomy (RNU) remains the cornerstone treatment, UTUC has high local and metastatic relapse rates, leading to a dismal prognosis. To identify the clinicopathological factors associated with an increased risk of local and metastatic relapse in UTUC, we conducted a retrospective analysis of 133 consecutive UTUC patients who underwent RNU from 1998 to 2018. Patients lost to follow-up or with a history of bladder cancer were excluded from the study. The remaining 87 patients were categorized into two subgroups: those with tumor recurrence/relapse (40 cases) and those without recurrence/relapse (47 cases). Clinical and pathological characteristics were compared across the two groups. Multiple factors are associated with UTUC recurrence/relapse including larger tumor size, histology divergent differentiations/subtypes, high tumor grade, advanced pathologic T stage, positive margin, lymphovascular invasion (LVI), positive lymph node status, and preoperative hydronephrosis. Multivariate Cox regression analysis revealed that squamous differentiation predicted recurrence/relapse (p = 0.012), independent of tumor stage. Moreover, compared to the conventional histology type, UTUC with squamous differentiation had a significantly higher relapse rate (p = 0.0001) and poorer survival (p = 0.0039). This observation was further validated in invasive high-grade UTUC cases. Our findings suggest that many pathological factors contribute to UTUC recurrence/relapse, particularly, squamous differentiation may serve as an independent risk predictor for relapse and a potent prognosticator for adverse cancer-specific survival in UTUC patients. Recognizing and thoroughly assessing the pathological factors is essential for better oncologic management of UTUC.

Pan-cancer analysis of prognostic metastatic phenotypes.

Although cancer is highly heterogeneous, all metastatic cancer is considered American Joint Committee on Cancer (AJCC) Stage IV disease. The purpose of this project was to redefine staging of metastatic cancer. Internal validation of nationally representative patient data from the National Cancer Database (n = 461 357; 2010-2013), and external validation using the Surveillance, Epidemiology and End Results database (n = 106 595; 2014-2015) were assessed using the concordance index for evaluation of survival prediction. A Cox proportional hazards model was used for overall survival by considering identified phenotypes (latent classes) and other confounding variables. Latent class analysis was performed for phenotype identification, where Bayesian information criterion (BIC) and sample-size-adjusted BIC were used to select the optimal number of distinct clusters. Kappa coefficients assessed external cluster validation. Latent class analysis identified five metastatic phenotypes with differences in overall survival (P < .0001): (Stage IVA) nearly exclusive bone-only metastases (n = 59 049, 12.8%; median survival 12.7 months; common in lung, breast and prostate cancers); (IVB) predominant lung metastases (n = 62 491, 13.5%; 11.4 months; common in breast, stomach, kidney, ovary, uterus, thyroid, cervix and soft tissue cancers); (IVC) predominant liver/lung metastases (n = 130 014, 28.2%; 7.0 months; common in colorectum, pancreatic, lung, esophagus and stomach cancers); (IVD) bone/liver/lung metastases predominant over brain (n = 61 004, 13.2%; 5.9 months; common in lung and breast cancers); and (IVE) brain/lung metastases predominant over bone/liver (n = 148 799, 32.3%; 5.7 months; lung cancer and melanoma). Long-term survivors were identified, particularly in Stages IVA-B. A pan-cancer nomogram model to predict survival (STARS: site, tumor, age, race, sex) was created, validated and provides 13% better prognostication than AJCC: 1-month concordance index of 0.67 (95% confidence interval [CI]: 0.66-0.67) vs 0.61 (95% CI: 0.60-0.61). STARS is simple, uses easily accessible variables, better prognosticates survival outcomes and provides a platform to develop novel metastasis-directed clinical trials.

Three-Dimensional Microtumors for Probing Heterogeneity of Invasive Bladder Cancer.

Bladder cancer is an increasingly common malignancy, and muscle invasive bladder cancer is associated with particularly high rates of morbidity and mortality. The morphologic and molecular diversity of bladder cancer poses significant challenges in elucidating the invasion mechanisms responsible for disease progression. Furthermore, conventional invasion assays do not provide a physiological context for studying bladder cancer invasion within 3D microenvironments and have limited ability to capture the contribution of cellular phenotypic heterogeneity to disease progression. Here, we describe the development of a 3D microtumor invasion model suitable for the analysis of cellular phenotypic heterogeneity in cell lines and primary tumor cells from bladder cancer patients. This model incorporates a self-assembly approach for recapitulating features of bladder cancer invasion in 3D microenvironments and probing the invasive cell subpopulations. The gene expression profiles of invading microtumors were analyzed by incorporating a gold nanorod-locked nucleic acid biosensor. The incorporation of the single cell biosensor and transient gene knockdown into the system revealed the formation of invasive leader cells with upregulated Delta-like ligand 4 (DLL4) expression as well as the role of NOTCH1-DLL4 signaling in collective bladder cancer invasion. The involvement of DLL4 expressing cells in bladder cancer invasion was also observed in patient samples obtained from transurethral resection. Collectively, our study demonstrates a 3D microtumor invasion model for investigating intracellular heterogeneity of bladder cancer invasion and analyzing patient derived samples toward personalized medicine applications.

Characterization of Histone Deacetylase Expression Within In Vitro and In Vivo Bladder Cancer Model Systems.

Epigenetic aberrations are prominent in bladder cancer (BC) and contribute to disease pathogenesis. We characterized histone deacetylase (HDAC) expression, a family of deacetylation enzymes, in both in vitro and in vivo BC model systems and analyzed expression data from The Cancer Genome Atlas (TCGA). Quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting analysis was used to determine the expression status of Class I and II HDACs in ten human BC cell lines, while qRT-PCR was used to determine HDAC expression in 24 human tumor specimens. The TCGA cohort consists of 408 muscle invasive BC (MIBC) clinical samples and analysis of this data set identified expression of HDAC4 and -9 as being associated with basal-squamous disease. These findings agree with qRT-PCR results identifying increased expression of HDAC4, -7, and -9 in basal BC cell lines (p < 0.05; Kruskal-Wallis test) and in clinical specimens with invasive bladder cancer (not statistically significant). We also observed increased expression in Hdac4, -7, and -9 in commonly used BC mouse models. Here, we identify suitable preclinical model systems for the study of HDACs, and show increased expression of Class IIa HDACs, specifically HDAC4 and HDAC9, in basal BC cell lines and in invasive clinical specimens. These results suggest this class of HDACs may be best suited for targeted inhibition in patients with basal BC.

Loss of FOXA1 Drives Sexually Dimorphic Changes in Urothelial Differentiation and Is an Independent Predictor of Poor Prognosis in Bladder Cancer.

We previously found loss of forkhead box A1 (FOXA1) expression to be associated with aggressive urothelial carcinoma of the bladder, as well as increased tumor proliferation and invasion. These initial findings were substantiated by The Cancer Genome Atlas, which identified FOXA1 mutations in a subset of bladder cancers. However, the prognostic significance of FOXA1 inactivation and the effect of FOXA1 loss on urothelial differentiation remain unknown. Application of a univariate analysis (log-rank) and a multivariate Cox proportional hazards regression model revealed that loss of FOXA1 expression is an independent predictor of decreased overall survival. An ubiquitin Cre-driven system ablating Foxa1 expression in urothelium of adult mice resulted in sex-specific histologic alterations, with male mice developing urothelial hyperplasia and female mice developing keratinizing squamous metaplasia. Microarray analysis confirmed these findings and revealed a significant increase in cytokeratin 14 expression in the urothelium of the female Foxa1 knockout mouse and an increase in the expression of a number of genes normally associated with keratinocyte differentiation. IHC confirmed increased cytokeratin 14 expression in female bladders and additionally revealed enrichment of cytokeratin 14-positive basal cells in the hyperplastic urothelial mucosa in male Foxa1 knockout mice. Analysis of human tumor specimens confirmed a significant relationship between loss of FOXA1 and increased cytokeratin 14 expression.

Mouse models of prostate cancer: picking the best model for the question.

When the National Institutes of Health Mouse Models of Human Cancer Consortium initiated the Prostate Steering Committee 15 years ago, there were no genetically engineered mouse (GEM) models of prostate cancer (PCa). Today, a PubMed search for "prostate cancer mouse model" yields 3,200 publications and this list continues to grow. The first generation of GEM utilized the newly discovered and characterized probasin promoter driving viral oncogenes such as Simian virus 40 large T antigen to yield the LADY and TRAMP models. As the PCa research field has matured, the second generation of models has incorporated the single and multiple molecular changes observed in human disease, such as loss of PTEN and overexpression of Myc. Application of these models has revealed that mice are particularly resistant to developing invasive PCa, and once they achieve invasive disease, the PCa rarely resembles human disease. Nevertheless, these models and their application have provided vital information on human PCa progression. The aim of this review is to provide a brief primer on mouse and human prostate histology and pathology, provide descriptions of mouse models, as well as attempt to answer the age old question: Which GEM model of PCa is the best for my research question?

FOXA1 deletion in luminal epithelium causes prostatic hyperplasia and alteration of differentiated phenotype.

The forkhead box (Fox) superfamily of transcription factors has essential roles in organogenesis and tissue differentiation. Foxa1 and Foxa2 are expressed during prostate budding and ductal morphogenesis, whereas Foxa1 expression is retained in adult prostate epithelium. Previous characterization of prostatic tissue rescued from embryonic Foxa1 knockout mice revealed Foxa1 to be essential for ductal morphogenesis and epithelial maturation. However, it is unknown whether Foxa1 is required to maintain the differentiated status in adult prostate epithelium. Here, we employed the PBCre4 transgenic system and determined the impact of prostate-specific Foxa1 deletion in adult murine epithelium. PBCre4/Foxa1(loxp/loxp) mouse prostates showed progressive florid hyperplasia with extensive cribriform patterning, with the anterior prostate being most affected. Immunohistochemistry studies show mosaic Foxa1 KO consistent with PBCre4 activity, with Foxa1 KO epithelial cells specifically exhibiting altered cell morphology, increased proliferation, and elevated expression of basal cell markers. Castration studies showed that, while PBCre4/Foxa1(loxp/loxp) prostates did not exhibit altered sensitivity in response to hormone ablation compared with control prostates, the number of Foxa1-positive cells in mosaic Foxa1 KO prostates was significantly reduced compared with Foxa1-negative cells following castration. Unexpectedly, gene expression profile analyses revealed that Foxa1 deletion caused abnormal expression of seminal vesicle-associated genes in KO prostates. In summary, these results indicate Foxa1 expression is required for the maintenance of prostatic cellular differentiation.

Deficiency in metabolic regulators PPARγ and PTEN cooperates to drive keratinizing squamous metaplasia in novel models of human tissue regeneration.

Hindgut-derived endoderm can differentiate into rectal, prostatic, and bladder phenotypes. Stromal-epithelial interactions are crucial for this development; however, the precise mechanisms by which epithelium responds to stromal cues remain unknown. We have previously reported ectopic expression of peroxisome proliferator-activated receptor-γ2 (PPARγ2) increased androgen receptor expression and promoted differentiation of mouse prostate epithelium. PPARγ is also implicated in urothelial differentiation. Herein we demonstrate that knockdown of PPARγ2 in benign human prostate epithelial cells (BHPrEs) promotes urothelial transdifferentiation. Furthermore, in vitro and in vivo heterotypic tissue regeneration models with embryonic bladder mesenchyme promoted urothelial differentiation of PPARγ2-deficient BHPrE cells, and deficiency of both PPARγ isoforms 1 and 2 arrested differentiation. Because PTEN deficiency is cooperative in urothelial pathogenesis, we engineered BHPrE cells with combined knockdown of PPARγ and PTEN and performed heterotypic recombination experiments using embryonic bladder mesenchyme. Whereas PTEN deficiency alone induced latent squamous differentiation in BHPrE cells, combined PPARγ and PTEN deficiency accelerated the development of keratinizing squamous metaplasia (KSM). We further confirmed via immunohistochemistry that gene expression changes in metaplastic recombinants reflected human urothelium undergoing KSM. In summary, these data suggest that PPARγ isoform expression provides a molecular basis for observations that adult human epithelium can be transdifferentiated on the basis of heterotypic mesenchymal induction. These data also implicate PPARγ and PTEN inactivation in the development of KSM.

Progress made in the use of animal models for the study of high-risk, nonmuscle invasive bladder cancer.

PURPOSE OF REVIEW: High-risk, nonmuscle invasive bladder cancer (HR-NMIBC) represents a costly and difficult-to-treat disease, the molecular pathogenesis of which has a limited understanding. Most preclinical models for the study of bladder cancer are more appropriate for the study of advanced disease. However, recent key advances in preclinical animal models places us at an opportune position to better understand HR-NMIBC. RECENT FINDINGS: Discoveries in the basic sciences allow us to better understand tumor biology when building models of bladder cancer. Of note, a key study on urothelial progenitor cells recently highlighted an important role for Sonic hedgehog-positive cells and retinoid signaling that is essential for urothelial development and regeneration. In the translational realm, transgenic mouse models continue to be used, with a recent interest in the role of Wnt/beta-catenin in urothelial carcinomas. Tissue recombination models are also being increasingly utilized to better recreate the tissue microenvironment and better understand stromal-epithelial interactions and the impact of genetic alterations on tissue differentiation. Lastly, the avatar mouse systems, which involve direct xenotransplantation of human tumor specimens into immunocompromised mice, represent an additional approach to study cancer characteristics in a preserved tissue context. SUMMARY: With molecular alterations remaining an unclear area of our understanding of HR-NMIBC, preclinical models of bladder cancer serve as essential tools to discover specific genetic compromises in disease pathogenesis and the therapeutics to treat them.

The transcription factor sex-determining region Y-box 2 (SOX2) in bladder cancer.

Sex-determining region Y-box 2 (SOX2) is a transcription factor with a central role in embryologic development. SOX2 is also an oncogene in several cancer types. Prior work by our group has shown SOX2 activity associates with cell cycle dysregulation in early-stage bladder cancer. The present study was thus undertaken to broadly investigate SOX2 in bladder cancer, with emphasis on associations with tumor stage, clinical outcomes, and tumorigenicity. Gene expression was quantified by immunohistochemistry in an established tissue microarray (n=303 cystectomy specimens, all stages) and whole tissue sections of noninvasive papillary urothelial carcinoma (n=25). Gene expression by RNA sequencing was evaluated in non-muscle invasive and muscle-invasive cohorts from publicly available repositories. By immunohistochemistry, SOX2 was expressed in 40% of whole tissue sections of noninvasive papillary carcinoma, which correlated with SOX2 expression by RNA sequencing (r=0.6, P=0.001, Spearman correlation). Expression tended to be focal (median H-score =6). SOX2 was expressed in only 9% of TMA cases, consistent with focal expression. SOX2 expression was substantially higher in muscle-invasive compared with noninvasive papillary urothelial carcinoma by RNA sequencing (P<0.001, Wilcoxon rank sum test). SOX2 expression associated with stage progression in lamina-propria invasive cancers (hazard ratio =2, P=0.05, Cox model, binary, RNA sequencing) but not noninvasive papillary cancers (P=0.5, Cox model, binary, RNA sequencing). SOX2 expression did not associate with overall survival in muscle-invasive carcinoma. Activity of SOX2 in bladder cancer was tested in vivo using murine allografts created with MB49 cells that express human SOX2 (MB49-SOX). MB49-SOX allografts expressed this protein focally by immunohistochemistry, much like human tumors. Compared with controls, MB49 allografts demonstrated larger tumor size (P=0.03, Wilcoxon rank sum test) and higher tumor burden in mesenteric metastases (P=0.009, Wilcoxon rank sum test). Though SOX2 expression is focal within tumors, it may drive tumorigenesis, increase growth rate, and promote aggressive features of bladder cancer, particularly stage progression of early-stage disease.

Macrophage migratory inhibitory factor promotes bladder cancer progression via increasing proliferation and angiogenesis.

Macrophage migratory inhibitory factor (MIF) is a proinflammatory cytokine shown to promote tumorigenesis. Using the N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) model of bladder cancer, we previously showed that MIF knockout mice display decreased angiogenesis and invasion compared with wild-type. This study examines the role of MIF in bladder cancer via use of oral inhibitors of MIF. In vitro, high-grade bladder cancer cells were treated with recombinant human MIF +/- (rhMIF+/-) inhibitor. Measurements included cell counts, proliferation by (3)H-thymidine incorporation (TdR), extracellular signal-regulated kinase (ERK) phosphorylation by western blot analysis, messenger RNA (mRNA) expression by quantitative PCR and protein secretion by enzyme-linked immunosorbent assay. Treatment with rhMIF increased ERK phosphorylation, cell counts, TdR and mRNA expression and protein secretion of vascular endothelial growth factor, which were blocked by specific inhibitors of ERK and MIF. In vivo, 3-month-old male C57Bl/6 mice were given BBN for 22 and 16 weeks in study 1 and study 2, respectively. Mice (n = 8-10 per group) were gavaged with vehicle or doses of MIF inhibitors daily from weeks 16-22 in both studies. Average bladder weights, reflecting tumor mass, tumor stage/burden, mitotic rate and proliferation indices, and microvessel densities were reduced in inhibitor groups versus controls. In summary, MIF promotes bladder cancer via increasing cell proliferation and angiogenesis and oral inhibitors of MIF may prove useful in treatment of this disease.

Histologic Analysis of Buccal Graft Quality Stratified by Tobacco Use in Patients Undergoing Substitution Urethroplasty.

OBJECTIVE: To determine if there are histologic differences relative to tobacco exposure in buccal mucosa. Substitution urethroplasty outcomes may be worse in tobacco users and we investigate if the buccal graft is inherently damaged due to chronic tobacco exposure. METHODS: Subjects undergoing substitution urethroplasty with buccal graft harvest were prospectively consented in this IRB approved study. Subjects with poor dentition were excluded. A detailed tobacco use history was obtained. Cotinine testing was performed day of surgery to confirm or exclude active tobacco use. Trimmed portions of harvested graft were sent for tissue processing. Standard hematoxylin and eosin staining was performed. A single blinded pathologist performed analysis of the slides. Using a scale of none, mild, moderate, or severe slides were analyzed for cytologic atypia, architectural complexity, inflammation, and keratinization. Evidence of vascular damage was noted and the type of inflammation if present was classified. RESULTS: Twenty-five buccal grafts were analyzed. No evidence of vascular damage or cytologic atypia were noted in any grafts. While mild architectural complexity and mild inflammation, typically lymphocytic, were noted in several of the buccal mucosa sections, this did not appear to correlate with tobacco exposure. The majority of grafts demonstrating increased keratinization correlated with significant tobacco exposure, but this was not consistently noted in all those with tobacco use. CONCLUSIONS: Buccal mucosa in patients with tobacco exposure did not show significant histologic alterations. Outcomes of substitution urethroplasty may be more impacted by persistent systemic exposure causing local ischemia as opposed to the graft tissue itself.

AUTS2 Controls Neuronal Lineage Choice Through a Novel PRC1-Independent Complex and BMP Inhibition.

Despite a prominent risk factor for Neurodevelopmental disorders (NDD), it remains unclear how Autism Susceptibility Candidate 2 (AUTS2) controls the neurodevelopmental program. Our studies investigated the role of AUTS2 in neuronal differentiation and discovered that AUTS2, together with WDR68 and SKI, forms a novel protein complex (AWS) specifically in neuronal progenitors and promotes neuronal differentiation through inhibiting BMP signaling. Genomic and biochemical analyses demonstrated that the AWS complex achieves this effect by recruiting the CUL4 E3 ubiquitin ligase complex to mediate poly-ubiquitination and subsequent proteasomal degradation of phosphorylated SMAD1/5/9. Furthermore, using primary cortical neurons, we observed aberrant BMP signaling and dysregulated expression of neuronal genes upon manipulating the AWS complex, indicating that the AWS-CUL4-BMP axis plays a role in regulating neuronal lineage specification in vivo. Thus, our findings uncover a sophisticated cellular signaling network mobilized by a prominent NDD risk factor, presenting multiple potential therapeutic targets for NDD.

Concurrent durvalumab and radiation therapy (DUART) followed by adjuvant durvalumab in patients with localized urothelial cancer of bladder: results from phase II study, BTCRC-GU15-023.

BACKGROUND: Patients with bladder cancer (BC) who are cisplatin ineligible or have unresectable disease have limited treatment options. Previously, we showed targeting programmed death-ligand 1 (PD-L1) with durvalumab (durva) and radiation therapy (RT) combination was safe in BC. We now report results from a phase II study evaluating the toxicity and efficacy of durva and RT in localized BC. METHODS: This is a single-arm, multi-institutional phase II study; N=26. Enrolled patients had pure or mixed urothelial BC (T2-4 N0-2 M0) with unresectable tumors and were unfit for surgery or cisplatin ineligible. Patients received durva concurrently with RT ×7 weeks, followed by adjuvant durva × 1 year. PRIMARY ENDPOINTS: (A) progression-free survival (PFS) at 1 year and (B) disease control rate (DCR) post adjuvant durva. Key secondary endpoints: (A) complete response (CR) post durvaRT (8 weeks), (B) overall survival (OS), (C) PFS and (D) toxicity. Correlative studies included evaluation of baseline tumor and blood (baseline, post durvaRT) for biomarkers. RESULTS: Median follow-up was 27 months. Evaluable patients: 24/26 post durvaRT, 22/26 for DCR post adjuvant durva, all patients for PFS and OS. Post adjuvant durva, DCR was seen in 72.7%, CR of 54.5%. 1-year PFS was 71.5%, median PFS was 21.8 months. 1-year OS was 83.8%, median OS was 30.8 months. CR at 8 weeks post durvaRT was 62.5%. Node positive (N+) patients had similar median PFS and OS. DurvaRT was well tolerated. Grade ≥3 treatment-related adverse events: anemia, high lipase/amylase, immune-nephritis, transaminitis, dyspnea (grade 4-COPD/immune), fatigue, rash, diarrhea and scleritis. No difference in outcome was observed with PD-L1 status of baseline tumor. Patients with CR/PR or SD had an increase in naïve CD4 T cells, a decrease in PD-1+CD4 T cells at baseline and an increase in cytokine-producing CD8 T cells, including interferon gamma (IFNγ) producing cells, in the peripheral blood. CONCLUSION: Durva with RT followed by adjuvant durva was safe with promising efficacy in localized BC patients with comorbidities, including N+ patients. Larger randomized studies, like S1806 and EA8185, are needed to evaluate the efficacy of combining immunotherapy and RT in BC. TRIAL REGISTRATION NUMBER: NCT02891161.

Characterization of laminin-332 gene expression in molecular subtypes of human bladder cancer.

Human bladder cancer (BCa) exhibits morphological and molecular heterogeneity which can complicate treatment. Morphologically, more than 90% of BCa is classified as urothelial cell carcinoma (UCC). Among other histological variants, UCC with squamous differentiation (SqD) shows a worse prognosis than pure UCC. In addition, basal-squamous BCa is enriched for SqD, and these tumors have a poor prognosis. Therefore, it is critical to elucidate the mechanisms to drive the basal-squamous phenotype of human BCa. Laminin-332 is a major glycoprotein of the epithelial basement membrane. It is well known that laminin-332 is a favorable target for extracellular matrix proteases such as matrix metalloproteinases (MMPs) in various diseases. Accumulating evidence indicates the significant role of laminin-332 in tumorigenesis. Here, we analyzed the expression of laminin-332 genes (LAMA3, LAMB3, LAMC2) in molecular subtypes of human BCa using publicly available data from The Cancer Genome Atlas (TCGA). Additionally, we also used q-RT-PCR to characterize laminin-332 gene expression between distinct molecular subtypes of human BCa cell lines. Our analysis of publicly available data show that laminin-332 genes are highly expressed in the basal-squamous molecular subtype of human BCa. In addition, we show laminin-332 genes are highly expressed in basal-squamous human BCa cell lines. Moreover, the expression of both LAMA3 and LAMC2 are negatively correlated with expression of the luminal transcription factor (TF) FOXA1 in the TCGA data. We also demonstrate that laminin-332 genes are downregulated by the overexpression of FOXA1 in a human basal-squamous BCa cell line (5637). Taken together, these results suggest that laminin-332 gene expression may be a biomarker of BCa patients with basal-squamous disease.

A transcriptional network of cell cycle dysregulation in noninvasive papillary urothelial carcinoma.

Human cancers display a restricted set of expression profiles, despite diverse mutational drivers. This has led to the hypothesis that select sets of transcription factors act on similar target genes as an integrated network, buffering a tumor's transcriptional state. Noninvasive papillary urothelial carcinoma (NIPUC) with higher cell cycle activity has higher risk of recurrence and progression. In this paper, we describe a transcriptional network of cell cycle dysregulation in NIPUC, which was delineated using the ARACNe algorithm applied to expression data from a new cohort (n = 81, RNA sequencing), and two previously published cohorts. The transcriptional network comprised 121 transcription factors, including the pluripotency factors SOX2 and SALL4, the sex hormone binding receptors ESR1 and PGR, and multiple homeobox factors. Of these 121 transcription factors, 65 and 56 were more active in tumors with greater and less cell cycle activity, respectively. When clustered by activity of these transcription factors, tumors divided into High Cell Cycle versus Low Cell Cycle groups. Tumors in the High Cell Cycle group demonstrated greater mutational burden and copy number instability. A putative mutational driver of cell cycle dysregulation, such as homozygous loss of CDKN2A, was found in only 50% of High Cell Cycle NIPUC, suggesting a prominent role of transcription factor activity in driving cell cycle dysregulation. Activity of the 121 transcription factors strongly associated with expression of EZH2 and other members of the PRC2 complex, suggesting regulation by this complex influences expression of the transcription factors in this network. Activity of transcription factors in this network also associated with signatures of pluripotency and epithelial-to-mesenchymal transition (EMT), suggesting they play a role in driving evolution to invasive carcinoma. Consistent with this, these transcription factors differed in activity between NIPUC and invasive urothelial carcinoma.

Avelumab in Combination with Eribulin Mesylate in Metastatic Urothelial Carcinoma: BTCRC GU-051, a Phase 1b Study.

BACKGROUND: Patients with metastatic urothelial carcinoma (mUC) have poor prognosis, so further development of novel combinations for these patients is needed. OBJECTIVE: To assess the safety and efficacy of eribulin mesylate (eribulin) with avelumab in mUC. DESIGN, SETTING, AND PARTICIPANTS: This was an open-label, phase 1b study in which patients with mUC who were cisplatin-ineligible and treatment-naïve or platinum-resistant were treated with eribulin and avelumab. A 3 + 3 design was used. The study was prematurely terminated because the free study drug became unavailable, but we performed extended follow-up for patients enrolled in the study. INTERVENTION: Patients received eribulin 1.1 mg/m2 plus avelumab 10 mg/kg on days 1 and 15 in every 28-d cycle in cohort 0, or eribulin 1.4 mg/m2 plus avelumab 10 mg/kg on days 1 and 15 in every 28-d cycle in cohort +1. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The primary objectives were to determine the maximum tolerated dose (MTD) of eribulin with avelumab and assess the objective response rate. A key secondary endpoint was to assess efficacy by evaluating the disease control rate. Exploratory endpoints included PD-1 expression on T cells in peripheral blood and in tumor cells, and tumor DNA sequencing. RESULTS AND LIMITATIONS: A total of six patients were enrolled in the MTD group (n = 3 in cohort 0 and n = 3 in cohort +1). No dose-limiting toxicity (DLT) was observed in cohort 0, whereas two DLT events were observed in cohort +1. Two patients in cohort 0 had a partial response that was durable, with one patient having a durable response for 7.8 mo. Disease control was observed in 4/6 patients (66.7%). Owing to the early termination, MTD could not be determined. CONCLUSIONS: While early termination of this trial precludes any definitive conclusions, the combination of eribulin and avelumab shows promise in mUC. We observed that treatment was better tolerated and efficacious at lower doses of eribulin. Further research is warranted for this combination in mUC. PATIENT SUMMARY: We evaluated different doses of eribulin (a chemotherapy drug) in combination with a fixed dose of avelumab (an antibody used to treat several different cancers) in a small group of patients with metastatic cancer of the urinary tract. The lower dose of eribulin was easier to tolerate and the combination had an anti-cancer effect. This trial is registered at ClinicalTrials.gov as NCT03502681.

FOXA1 repression drives lineage plasticity and immune heterogeneity in bladder cancers with squamous differentiation.

Cancers arising from the bladder urothelium often exhibit lineage plasticity with regions of urothelial carcinoma adjacent to or admixed with regions of divergent histomorphology, most commonly squamous differentiation. To define the biologic basis for and clinical significance of this morphologic heterogeneity, here we perform integrated genomic analyses of mixed histology bladder cancers with separable regions of urothelial and squamous differentiation. We find that squamous differentiation is a marker of intratumoral genomic and immunologic heterogeneity in patients with bladder cancer and a biomarker of intrinsic immunotherapy resistance. Phylogenetic analysis confirms that in all cases the urothelial and squamous regions are derived from a common shared precursor. Despite the presence of marked genomic heterogeneity between co-existent urothelial and squamous differentiated regions, no recurrent genomic alteration exclusive to the urothelial or squamous morphologies is identified. Rather, lineage plasticity in bladder cancers with squamous differentiation is associated with loss of expression of FOXA1, GATA3, and PPARG, transcription factors critical for maintenance of urothelial cell identity. Of clinical significance, lineage plasticity and PD-L1 expression is coordinately dysregulated via FOXA1, with patients exhibiting morphologic heterogeneity pre-treatment significantly less likely to respond to immune checkpoint inhibitors.

Intratumoral Heterogeneity Promotes Collective Cancer Invasion through NOTCH1 Variation.

Cellular and molecular heterogeneity within tumors has long been associated with the progression of cancer to an aggressive phenotype and a poor prognosis. However, how such intratumoral heterogeneity contributes to the invasiveness of cancer is largely unknown. Here, using a tumor bioengineering approach, we investigate the interaction between molecular subtypes within bladder microtumors and the corresponding effects on their invasiveness. Our results reveal heterogeneous microtumors formed by multiple molecular subtypes possess enhanced invasiveness compared to individual cells, even when both cells are not invasive individually. To examine the molecular mechanism of intratumoral heterogeneity mediated invasiveness, live single cell biosensing, RNA interference, and CRISPR-Cas9 gene editing approaches were applied to investigate and control the composition of the microtumors. An agent-based computational model was also developed to evaluate the influence of NOTCH1 variation on DLL4 expression within a microtumor. The data indicate that intratumoral variation in NOTCH1 expression can lead to upregulation of DLL4 expression within the microtumor and enhancement of microtumor invasiveness. Overall, our results reveal a novel mechanism of heterogeneity mediated invasiveness through intratumoral variation of gene expression.